MOLECULAR CHARACTERIZATION AND OVEREXPRESSION OF THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE GENE FROM TRYPANOSOMA-CRUZI

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme in T rypanosoma cruzi is a rational target for the treatment of Chagas dise ase. To evaluate the T. cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T. cruzi DNA and sequenced. Trans lation of the nucleotide sequence of the hgprt revealed an open readin g frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino aci ds. The T. cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid seq uence identity to its human, Plasmodium falciparum, and Schistosoma ma nsoni counterparts, respectively, but was 50% identical to the T. bruc ei HGPRT protein. Northern analysis of T. cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that h gprt was a single copy gene within the T. cruzi genome. The T. cruzi h gprt was inserted into the pBAce expression plasmid and transformed in to Escherichia coil that are deficient in hypoxanthine and guanine pho sphoribosylating activities. High levels of soluble, enzymatically act ive T. cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies. The recombinant HGP RT was purified to apparent homogeneity by GTP-agarose affinity chroma tography and recognized hypoxanthine, guanine, and allopurinol, but no t adenine or xanthine, as substrates. The availability of the hgprt cl one and large amounts of pure HGPRT protein provide a foundation for a structure-based drug design strategy for the treatment of Chagas dise ase.